Glutamine Synthetase (GS) is a plant enzyme which has a central role in the assimilation of ammonia and in the regulation of nitrogen metabolism. Since in most plants glutamine synthetase is, via the glutamine synthetase/glutamate synthase pathway (FIG. 1), the only efficient way to detoxify ammonia released by nitrate reduction, amino acid degradation or photorespiration, plants are very susceptible to potent inhibitors of glutamine synthetase.
One of the most potent glutamine synthetase inhibitors known at present is phosphinothricin (1) (hereinafter PPT): ##STR1##
PPT is a glutamic acid analogue. The compound was initially isolated from a tripeptide antibiotic produced by Streptomyces viridochromogenes (Bayer, E., et al., Helvetica Chimica Acta 55:224 (1972), see also German Patent DOS 2717440, Hoechst, A.G.). PPT is a potent competitive inhibitor of glutamine synthetase from E. coli with a K.sub.i of 0.0059 mM.
Schwerdtle, F. (Zeitschrift fur Pflanzen-Krankheiten und Pflanzenschutz, IX:431-440 (1981)) demonstrated that PPT is a non-selective foliar herbicide for the control of undesirable mono and dicotyledonous plants in orchards, vineyards, plantations with minimum tillage, direct drilling, and as a harvest aid. Field trials in West Germany, Spain, South Africa, U.S.A. and Japan showed that most dicotyledonous weeds were well controlled. For monocotyledonous weeds somewhat higher quantities were needed for good control. Leason, M., et al. (Phytochemistry 21:855-857 (1982)) demonstrated that PPT is a mixed competitive inhibitor of pea leaf glutamine synthetase with an apparent K.sub.i value of 0.073 mM.
It would be of great interest to be able to confer resistance to PPT, as well as to other GS inhibitors to selected plans, since herbicidal selectivity is quite crucial in any commercially useful herbicide. PPT, as indicated, is non-selective.
There is some precedent for the existence of glutamine synthetases resistant to other compounds. It is known that methionine sulfoximine (MSO), another glutamate analogue, is a mixed competitive inhibitor (K.sub.i value of 0.16 mM) of pea leaf glutamine synthetase (Leason, M., et al., Phytochemistry 21:855-857 (1982)). Miller, E. S. and Brenchley, J. E. (The Journal of Bioloqical Chemistry 256:11307-11321 (1981)) studied the properties of several mutants of Salmonella resistant to MSO. One mutation apparently altered glutamine synthetase at the ammonia binding domain, conferring MSO resistance. More recent, Young and Ringold (ibid 258:11260-11266 (1983)) have reported that mouse 3T6 cells grown in the presence of MSO developed resistance thereto. MSO resistant cells had mRNA enriched for glutamine synthetase, and the authors suggested that this observation implied an amplification of the gene. See also Sanders and Wilson, EMBO. J. 3:65-71 (1984). Neither the Miller, Young, nor Sanders studies were reported on plant GS.
Further, prior to the present invention no studies have been reported on attempts to confer resistance to herbicidal GS inhibitors, such as PPT, by manipulating the glutamine synthetase genes in plant cells.
It would therefore be desirable to develop plant cells which are resistant to herbicidal inhibitors of GS, such as PPT, by manipulating the plant glutamine synthetase genes in said cells. In such manner, it would be possible to confer herbicidal selectivity to any given plant.